Laser line illuminator for high throughput sequencing

ABSTRACT

Imaging systems including an objective lens and a line generation module are described herein. The objective lens may focus a first light beam emitted by the line generation module and a second light beam emitted by the line generation module at a focal point external to a sample so as to adjust line width. Line width may be increased to lower overall power density of a light beam on a surface of the sample such that the power density of the light beam on the surface of the sample is below a photosaturation threshold of a dye on the sample.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional PatentApplication No. 62/468,883 filed on Mar. 8, 2017 and titled “Laser LineIlluminator For High Throughput Sequencing,” which is incorporatedherein by reference in its entirety. The present application also claimsthe benefit of Netherlands Patent Application No. N2018855 filed on May5, 2017, and titled “Laser Line Illuminator For High ThroughputSequencing.”

BACKGROUND

Biological optical analysis instruments, such as genetic sequencers,tend to include multiple configurable components, each with multipledegrees of freedom. Increasing complexity of these biological opticalanalysis instruments has led to increased manufacturing and operationexpense. Generally, these types of instruments benefit from precisealignment of their many internal optical components. In some geneticsequencing instruments, for example, internal components are generallyaligned within precise tolerances. Many manufacturing techniques forsuch instruments involve installing all of the components on a precisionplate, and then configuring and aligning each component. Componentalignment may change during shipping or use. For example, temperaturechanges may alter alignments. Re-aligning each component takes time andskill. In some examples, there may be over 30 total degrees of freedomavailable across all of the components and they interact to each other.The large number of degrees of freedom complicates alignment andconfiguration and adds time and expense to system operation. Opticalsequencer fabrication and operation may be simplified by reducing thedegrees of freedom available across all system components through amodular architecture.

Optical sequencers may use laser line illumination to detect andsequence a biological specimen. For example, laser line illumination mayenable high throughput scanning using a time delay integration (TDI)sensor to detect fluorescence emissions from a sample flowcell. Thedetected emissions may be used to identify and sequence geneticcomponents of the biological sample. However, at high scanning speedsand/or laser output powers, functionality may be impacted byphoto-saturation of the fluorophores and/or photo-bleaching of thefluorophores, and/or photo-induced damage to the sample. High powerlasers can also cause damage to the objective lens, including thebonding adhesive, coatings and glass.

SUMMARY

Various implementations of the technologies disclosed herein describeimaging systems including an objective lens and a line generationmodule, where the imaging system is configured to adjust the width oflines emitted by the line generation module on a surface of a biologicalsample.

In one example, an imaging system includes: a line generation module andan objective lens. The line generation module includes a first lightsource to emit a first light beam at a first wavelength; a second lightsource to emit a second light beam at a second wavelength; and one ormore line forming optics to shape a light beam emitted by the firstlight source into a line and a light beam emitted by the second lightsource into a line. In this example, the objective is configured tofocus the first light beam and the second light beam at a focal pointexternal to a sample of a sampling structure.

In one example, the sampling structure includes: a cover plate asubstrate, and a liquid passage between the cover plate and substrate.In this example, the liquid passage includes a top interior surface anda bottom interior surface, and the sample is located at the top interiorsurface or at the bottom interior surface of the liquid passage. Thefocal point may be below the bottom interior surface of the liquidpassage as to increase a line width of the first light beam and a linewidth of the second line beam at the top interior surface of thesampling structure. Alternatively, the focal point may be above thebottom interior surface of the liquid passage as to increase a linewidth of the first light beam and a line width of the second line beamat the top interior surface of the sampling structure.

In some implementations, the sampling structure is detachably coupled tothe imaging system. In a particular implementation, the samplingstructure is a flowcell.

In particular implementations, the focal point is between about 50 μmand about 150 μm below the bottom interior surface of the samplingstructure. Alternatively, the focal point is between about 50 μm andabout 150 μm above the bottom interior surface of the samplingstructure.

In one implementation the imaging system includes a time delayintegration (TDI) sensor to detect fluorescence emissions from thesample. In particular implementations, the TDI sensor has a pixel sizebetween about 5 μm and about 15 μm, a sensor width between about 0.4 mmand about 0.8 mm, and a sensor length between about 16 mm and about 48mm.

In one implementation, the line width of the first light beam and theline width of the second light beam is between about 10 μm and about 30μm. In another implementation, the line length of the first light beamand the line length of the second light is between about 1 mm and about1.5 mm.

In one implementation, the one or more line widening optics include adefocus lens, a prism, or a diffuser. In a particular implementation,the one or more line widening optics include a Powell lens positionedafter a defocus lens in an optical path from the light sources to theobjective lens.

In some implementations, the line width of the first light beam isincreased to lower overall power density of the first light beam on asurface of the sample such that the power density of the first lightbeam on the surface of the sample is below a photosaturation thresholdof a first dye on the sample, and the line width of the second lightbeam is increased to lower overall power density of the second lightbeam on a surface of the sample such that the power density of thesecond light beam on the surface of the sample is below aphotosaturation threshold of a second dye on the sample.

In some implementations, the imaging system includes a z-stage forarticulating the objective to adjust the line width of the first lightbeam and to adjust the line width of the second light beam. In furtherimplementations, the imaging system includes a processor; and anon-transitory computer readable medium with computer executableinstructions embedded thereon, the computer executable instructionsconfigured to cause the system to: determine a quality of a signal fromthe TDI sensor; and articulate the objective in the z-axis to adjust thefocal point and optimize the quality of the signal from the TDI sensor.

In another example, a DNA sequencing system includes: a line generationmodule and an objective lens. In this example, the line generationmodule may include: a plurality of light sources, each light sourcebeing to emit a light beam; and one or more line forming optics to shapeeach light beam into a line; and the objective lens or the one or moreline forming optics are to increase a width of each line at a firstsurface or a second surface of a flowcell.

In implementations of this example, the objective lens is to focus eachlight beam at a focal point external to an interior surface of theflowcell as to increase the width of each line at the first surface orthe second surface of the flowcell. The focal point may be between about50 μm and about 150 μm below the bottom interior surface of the flowcellor between about 50 μm and about 150 μm above the top interior surfaceof the flowcell.

In some implementations, an objective lens of the imaging system isdesigned to be slightly finite conjugate to focus collimated laser lighta distance between about 50 and about 150 um below the imaged surface.

In some implementations, the line generation module (LGM) providesuniform line illumination at a desired aspect ratio using a Powell lens,or other beam shaping optics. The system may be configured to opticallyadjust the diffractive limited focal point on objective plane (e.g., theflowcell surfaces. By adjusting the focal point above or below thesurfaces of the flowcell, the beam width incident on the surfaces of theflowcell may be increased, and the laser power intensity at the sampleand the flowcell may be decreased. The power density may be controlledbelow or near photosaturation of fluorophores for genetic sampledetection (e.g., DNA, RNA, or other sample detection), while stillsatisfying TDI sensor integration tolerances on noise and speed. In someimplementations, grouping components of a modular optical analyticsystem into modular sub-assemblies, and then installing the modularsub-assemblies on a precision plate or other stable structure may reducerelative degrees of freedom and simplify overall system maintenance.

For example, in one implementation, a modular optical analytic systemmay include sets of components grouped into four modular subassemblies.A first modular subassembly may include a plurality of lasers andcorresponding laser optics grouped together into an LGM. A secondmodular subassembly may include lenses, tuning and filtering opticsgrouped into an emission optics module (EOM). A third modularsubassembly may include camera sensors and corresponding optomechanicsgrouped into a camera module (CAM). A fourth modular subassembly mayinclude focus tracking sensors and optics grouped into a focus trackingmodule (FTM). In some implementations, components of the system maygroup into different modular subassemblies. Components may be groupedinto fewer or greater numbers of subassemblies depending on the specificapplication and design choices. Each modular subassembly may bepre-fabricated by incorporating the individual components onto amounting plate or enclosure and precisely aligning and configuring thecomponents within the modular sub-assembly to predetermined tolerances.Each modular sub-assembly may be fabricated to minimize degrees offreedom, such that only key components may be moved in one or moredirections, or rotated, to enable precision alignment.

In some implementations, the LGM may be preconfigured on an LGM assemblybench designed with a precision interface and optics. The LGM assemblybench may include an assembly objective lens, a beam profiler, alignmenttargets, attenuator, precision plate, and translation stages. Theassembly objective lens may have a field of view, focal length, and workdistance that are greater than that of the EOM on the modular opticssystem, as to enable initial alignment of the laser modules and internaloptics of the LGM. The beam profiler may be a 2D imaging sensorconfigured to detect and report beam intensity at various targetlocations. Alignment of the beams may include optimizing the beamposition, intensity, pointing direction at these target locations bymanipulating various internal optics and/or mirrors within the LGM. Themanipulation of the various internal optical components and theevaluation of the laser using the beam profiler, may be an automatedprocess or a manual process.

The system may also include a precision mounting plate. The precisionmounting plate may be fabricated with alignment surfaces, such asmounting pins, grooves, slots, grommets, tabs, magnets, datum surfaces,tooling balls, or other surfaces designed to accept and mount eachpre-fabricated and tested modular subassembly in its desired position.The precision mounting plate may include flat structures, non-flatstructures, solid structures, hollow structures, honeycombed or latticedstructures, or other types of rigid mounting structures as known in theart. In some examples, the precision mounting plate incorporates or iscoupled to a stage motion assembly configured to maintain a levelmounting surface and dampen vibration. The stage assembly may includeactuators to control one or more control surfaces of an optical targetto provide feedback to align the modular subassemblies (e.g., the EOMand CAM), for example, to reposition one or more optical components orsensors within predetermined tolerances. These precision motion devicesmay accurately position the illumination lines within the field of viewof the optical imaging system, in stepwise or continuous motions.

Assembling a modular optical analytic system may include mounting eachmodular sub-assembly on the precision mounting plate and performing afinal alignment using one or more control adjustments. In some examples,an optical analytic system with more than 30 degrees of freedom acrosseach of its components may be reduced to a modular optical analyticsystem with fewer than 10 degrees of freedom across each of itscomponents, wherein the components are grouped into pre-configuredmodular subassemblies. These remaining degrees of freedom may beselected to optimize inter-component alignment tolerances withoutimplementing active or frequent alignment processes. In someimplementations, one or more control adjustments within one or moremodular subassemblies may be actuated using one or more correspondingactuators mounted in the subassemblies.

Sensors and/or detectors within one or more of the modular subassemblies(e.g., the CAM or the FTM) may be configured to transmit data to acomputer, the computer including a processor and non-transitory computerreadable media with machine-readable instructions stored thereon. Thesoftware may be configured to monitor optimal system performance, forexample, by detecting and analyzing beam focus, intensity, and shape. Insome examples, the system may include an optical target configured todisplay patterns specific to the alignment and performance of eachmodular subassembly. The software may then indicate via a graphical userinterface when a particular modular subassembly is operatingsub-optimally and recommend an open loop adjustment or implement acourse of closed loop action to rectify the issue. For example, thesoftware may be configured to transmit signals to the actuators toreposition specific components within predetermined tolerances, or maysimply recommend swapping out the under-performing modular sub-assembly.The software may be operated locally or remotely via a networkinterface, enabling remoted system diagnostics and tuning.

Other features and aspects of the disclosed technology will becomeapparent from the following detailed description, taken in conjunctionwith the accompanying drawings, which illustrate, by way of example, thefeatures in accordance with examples of the disclosed technology. Thesummary is not intended to limit the scope of any inventions describedherein, which are defined by the claims and equivalents.

It should be appreciated that all combinations of the foregoing concepts(provided such concepts are not mutually inconsistent) are contemplatedas being part of the inventive subject matter disclosed herein. Inparticular, all combinations of claimed subject matter appearing at theend of this disclosure are contemplated as being part of the inventivesubject matter disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The technology disclosed herein, in accordance with one or moreimplementations, is described in detail with reference to the followingfigures. These figures are provided to facilitate the reader'sunderstanding of the disclosed technology, and are not intended to beexhaustive or to limit the disclosure to the precise forms disclosed.Indeed, the drawings in the figures are provided for purposes ofillustration only, and merely depict typical or example implementationsof the disclosed technology. Furthermore, it should be noted that forclarity and ease of illustration, the elements in the figures have notnecessarily been drawn to scale.

FIG. 1A illustrates a generalized block diagram of an example imagescanning system with which systems and methods disclosed herein may beimplemented.

FIG. 1B is a perspective view diagram illustrating an example modularoptical analytic system in accordance with implementations disclosedherein.

FIG. 1C is a perspective view diagram illustrating an example precisionmounting plate in accordance with implementations disclosed herein.

FIG. 1D illustrates a block diagram of an example modular opticalanalytic system consistent with implementations disclosed herein.

FIG. 1E illustrates a perspective view of an example modular opticsanalytic system, consistent with implementations disclosed herein.

FIG. 1F illustrates a block diagram of a line generation module (LGM)alignment system, consistent with implementations disclosed herein.

FIG. 1G illustrates a perspective view of an LGM alignment system,consistent with implementations disclosed herein.

FIG. 1H illustrates a top down view of an example modular opticalanalytic system consistent with implementations disclosed herein.

FIG. 1I illustrates a side view of an example modular optical analyticsystem consistent with implementations disclosed herein.

FIG. 1J illustrates a block diagram of an LGM, an objective lens, andflowcell, consistent with implementations disclosed herein.

FIG. 1K illustrates a block diagram of an LGM and EOM system used todefocus the laser line pattern on a flowcell to avoid photo-saturationand photo-bleaching, consistent with implementations disclosed herein.

FIG. 2A is a side view diagram illustrating an emission optical module(EOM) in accordance with implementations disclosed herein.

FIG. 2B is a top-down diagram illustrating an EOM in accordance withimplementations disclosed herein.

FIG. 3A is a back view diagram illustrating a focus tracking module(FTM) in accordance with implementations disclosed herein.

FIG. 3B is a side view diagram illustrating an FTM in accordance withimplementations disclosed herein.

FIG. 3C is a top-down view diagram illustrating an FTM in accordancewith implementations disclosed herein.

FIG. 4A is a side view diagram illustrating an example modular opticalanalytic system in accordance with implementations disclosed herein.

FIG. 4B is a block diagram illustrating an example configuration for atube lens subassembly from an EOM, in accordance with implementationsdisclosed herein.

FIG. 4C is a block diagram illustrating another example configurationfor a tube lens subassembly from an EOM, in accordance withimplementations disclosed herein.

FIG. 5A is a side view diagram illustrating an FTM and an EOM withimplementations disclosed herein.

FIG. 5B is a top-down view diagram illustrating an example FTM and anEOM in accordance with implementations disclosed herein.

FIG. 6 is a side view diagram illustrating a line generation module(LGM) and an EOM in accordance with implementations disclosed herein.

FIG. 7 is a top-down view diagram illustrating an LGM and an EOM inaccordance with implementations disclosed herein.

FIG. 8 is a diagram illustrating an example process for installing andconfiguring a modular optical analytic system in accordance withimplementations disclosed herein.

FIG. 9 illustrates an example computing engine that may be used inimplementing various features of implementations of the disclosedtechnology.

It should be understood that the disclosed technology can be practicedwith modification and alteration, and that the disclosed technology belimited only by the claims and the equivalents thereof.

DETAILED DESCRIPTION

As used herein, the term “xy plane” is intended to mean a 2 dimensionalarea defined by straight line axes x and y (according to the Cartesiancoordinate system). When used in reference to a detector and an objectobserved by the detector, the area can be further specified as beingorthogonal to the direction of observation between the detector andobject being detected. When used herein to refer to a line scanner, theterm “y direction” refers to the direction of scanning.

As used herein, the term “z direction” or “z axis” is intended tospecify a direction or axis that is orthogonal to an area of an objectthat is observed by a detector. For example, the direction of focus foran optical system may be specified along the z axis.

Some implementations disclosed herein provide a modular optical system,such as ones that may be used for analyzing biological samples. Otherimplementations disclosed herein provide methods for assembling andinstalling modular optical systems for analyzing biological samples. Onesuch optical system may be, or may be part of a genomic sequencinginstrument. The instrument may be used to sequence DNA, RNA, or otherbiological samples. Some genomic sequencing instruments operate byfocusing coherent or incoherent light sources operating at differentwavelengths through internal optics and onto the sample. Base pairspresent in the sample then fluoresce and return light through the opticsof the sequencer and onto an optical sensor, which can then detect thetypes of base pairs present. These types of instruments rely on precisealignment and tuning of the internal optics and are sensitive todrifting or misalignment of components caused by thermal effects (e.g.,by heat from the light sources and electronics), as well as mechanicaleffects such as vibrations or incidental contact from users.Implementations of the present disclosure address these problems, andthe installation and maintenance costs associated therewith, through amodular approach. Groupings of functionally related optical componentsmay be pre-packaged, tested, and aligned as modular subassemblies. Eachmodular subassembly then may be treated as a field replaceable unit(FRU) which may be installed and aligned to the other modularsubassemblies in the system by mounting the subassembly to a precisionalignment plate.

Some implementations of the disclosure provide a system including aplurality of modular subassemblies and a precision mounting plate or,wherein each modular subassembly includes an enclosure and a pluralityof optical components aligned to the enclosure. The enclosure mayinclude a plurality of precision mounting structures, and each modularsubassembly may be mechanically coupled to the precision mounting plate,such that each precision mounting structure from a modular subassemblyattaches directly to a corresponding precision mounting structurelocated on the precision mounting plate or an adjacent modularsubassembly. In some examples, the line generation module includes afirst light source operating at a first wavelength, a second lightsource operating at a second wavelength, and a beam shaping lens alignedat a predetermined angle to each light source. For example, the firstwavelength may be a green wavelength and the second wavelength may be ared wavelength. The beam shaping lens may be a Powell lens.

In some implementations, the emissions optics module may include anobjective lens that is optically coupled to a light generation module,and a tube lens that is optically coupled to the objective lens. Theobjective lens focuses light onto a flowcell positioned at apredetermined distance from the flowcell. The objective may articulatealong a longitudinal axis, and the tube lens may include a lenscomponent that also articulates along a longitudinal axis within thetube lens to ensure accurate imaging. For example, the lens componentmay move to compensate for spherical aberration caused by articulationof the objective to image one or more surfaces of the flowcell.

In some examples, the flowcell may include a translucent cover plate, asubstrate, and a liquid sandwiched therebetween, and a biological samplemay be located at an inside surface of the translucent cover plate or aninside surface of the substrate. For example, the biological sample mayinclude DNA, RNA, or another genomic material which may be sequenced.

The focus tracking module may include a focus tracking light source anda focus tracking sensor, wherein the light source may generate a lightbeam, transmit the light beam through the plurality of opticalcomponents such that the light beam terminates at the focus trackingsensor. The focus tracking sensor may be communicatively coupled to aprocessor and a non-transitory computer readable medium withmachine-readable instructions stored thereon. The machine-readableinstructions, when executed, may cause the processor to receive anoutput signal from the focus tracking sensor and analyze the outputsignal to determine a set of characteristics of the light beam. In someexamples, the machine-readable instructions, when executed, furthercause the processor to generate a feedback signal indicating that one ormore of the optical components should be reconfigured to optimize theset of characteristics of the light beam. One or more of the modularsubassemblies may be a field replaceable unit. The precision mountingstructures may include a slot, a datum, a tab, a pin, or a recessedcavity, other mechanical mounting structures as known in the art, or anycombination thereof.

In some examples, the camera module includes a plurality of opticalsensors, and the light generation module includes a plurality of lightsources, wherein each optical sensor may be oriented to receive anddetect a light beam from corresponding light source.

Before describing various implementations of the systems and methodsdisclosed herein, it is useful to describe an example environment withwhich the systems and methods can be implemented. One such exampleenvironment is that of an optical system, such as that illustrated inFIG. 1A. The example optical system may include a device for obtainingor producing an image of a region. The example outlined in FIG. 1 showsan example imaging configuration of a backlight design implementation.

As can be seen in the example of FIG. 1A, subject samples are located onsample structure or container 110 (e.g., a flowcell as disclosedherein), which is positioned on a sample stage 170 under an objectivelens. Light source 160 and associated optics direct a beam of light,such as laser light, to a chosen sample location on the sample container110. The sample fluoresces and the resultant light is collected by theobjective lens and directed to a photo detector 140 to detect theflorescence. Sample stage 170 is moved relative to objective lens toposition the next sample location on sample container 110 at the focalpoint of the objective lens. Movement of sample stage 170 relative toobjective lens can be achieved by moving the sample stage itself, theobjective lens, the entire optical stage, or any combination of theforegoing. Further implementations may also include moving the entireimaging system over a stationary sample.

Fluid delivery module or device 100 directs the flow of reagents (e.g.,fluorescent nucleotides, buffers, enzymes, cleavage reagents, etc.) to(and through) sample container 110 and waste valve 120. In particularimplementations, the sample container 110 can be implemented as aflowcell that includes clusters of nucleic acid sequences at a pluralityof sample locations on the sample container 110. The samples to besequenced may be attached to the substrate of the flowcell, along withother optional components.

The system also comprises temperature station actuator 130 andheater/cooler 135 that can optionally regulate the temperature ofconditions of the fluids within the sample container 110. Camera system140 can be included to monitor and track the sequencing of samplecontainer 110. Camera system 140 can be implemented, for example, as aCCD camera, which can interact with various filters within filterswitching assembly 145, objective lens, and focusing laser/focusinglaser assembly 150. Camera system 140 is not limited to a CCD camera andother cameras and image sensor technologies can be used.

Light source 160 (e.g., an excitation laser within an assemblyoptionally comprising multiple lasers) or other light source can beincluded to illuminate fluorescent sequencing reactions within thesamples via illumination through fiber optic interface (which canoptionally comprise one or more re-imaging lenses, a fiber opticmounting, etc). Low watt lamp 165, focusing laser 150, and reversedichroic are also presented in the example shown. In someimplementations focusing laser 150 may be turned off during imaging. Inother implementations, an alternative focus configuration can include asecond focusing camera (not shown), which can be a quadrant detector, aPosition Sensitive Detector (PSD), or similar detector to measure thelocation of the scattered beam reflected from the surface concurrentwith data collection.

Although illustrated as a backlit device, other examples may include alight from a laser or other light source that is directed through theobjective lens onto the samples on sample container 110. Samplecontainer 110 can be ultimately mounted on a sample stage 170 to providemovement and alignment of the sample container 110 relative to theobjective lens. The sample stage can have one or more actuators to allowit to move in any of three dimensions. For example, in terms of theCartesian coordinate system, actuators can be provided to allow thestage to move in the X, Y and Z directions relative to the objectivelens. This can allow one or more sample locations on sample container110 to be positioned in optical alignment with objective lens.

A focus (z-axis) component 175 is shown in this example as beingincluded to control positioning of the optical components relative tothe sample container 110 in the focus direction (typically referred toas the z axis, or z direction). Focus component 175 can include one ormore actuators physically coupled to the optical stage or the samplestage, or both, to move sample container 110 on sample stage 170relative to the optical components (e.g., the objective lens) to provideproper focusing for the imaging operation. For example, the actuator maybe physically coupled to the respective stage such as, for example, bymechanical, magnetic, fluidic or other attachment or contact directly orindirectly to or with the stage. The one or more actuators can beconfigured to move the stage in the z-direction while maintaining thesample stage in the same plane (e.g., maintaining a level or horizontalattitude, perpendicular to the optical axis). The one or more actuatorscan also be configured to tilt the stage. This can be done, for example,so that sample container 110 can be leveled dynamically to account forany slope in its surfaces.

Focusing of the system generally refers to aligning the focal plane ofthe objective lens with the sample to be imaged at the chosen samplelocation. However, focusing can also refer to adjustments to the systemto obtain a desired characteristic for a representation of the samplesuch as, for example, a desired level of sharpness or contrast for animage of a test sample. Because the usable depth of field of the focalplane of the objective lens may be small (sometimes on the order of 1 μmor less), focus component 175 closely follows the surface being imaged.Because the sample container is not perfectly flat as fixtured in theinstrument, focus component 175 may be set up to follow this profilewhile moving along in the scanning direction (herein referred to as they-axis).

The light emanating from a test sample at a sample location being imagedcan be directed to one or more detectors 140. Detectors can include, forexample a CCD camera. An aperture can be included and positioned toallow only light emanating from the focus area to pass to the detector.The aperture can be included to improve image quality by filtering outcomponents of the light that emanate from areas that are outside of thefocus area. Emission filters can be included in filter switchingassembly 145, which can be selected to record a determined emissionwavelength and to cut out any stray laser light.

In various implementations, sample container 110 can include one or moresubstrates upon which the samples are provided. For example, in the caseof a system to analyze a large number of different nucleic acidsequences, sample container 110 can include one or more substrates onwhich nucleic acids to be sequenced are bound, attached or associated.In various implementations, the substrate can include any inertsubstrate or matrix to which nucleic acids can be attached, such as forexample glass surfaces, plastic surfaces, latex, dextran, polystyrenesurfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces,and silicon wafers. In some applications, the substrate is within achannel or other area at a plurality of locations formed in a matrix orarray across the sample container 110.

Although not illustrated, a controller can be provided to control theoperation of the scanning system. The controller can be implemented tocontrol aspects of system operation such as, for example, focusing,stage movement, and imaging operations. In various implementations, thecontroller can be implemented using hardware, algorithms (e.g., machineexecutable instructions), or a combination of the foregoing. Forexample, in some implementations the controller can include one or moreCPUs or processors with associated memory. As another example, thecontroller can comprise hardware or other circuitry to control theoperation, such as a computer processor and a non-transitory computerreadable medium with machine-readable instructions stored thereon. Forexample, this circuitry can include one or more of the following: fieldprogrammable gate array (FPGA), application specific integrated circuit(ASIC), programmable logic device (PLD), complex programmable logicdevice (CPLD), a programmable logic array (PLA), programmable arraylogic (PAL) or other similar processing device or circuitry. As yetanother example, the controller can comprise a combination of thiscircuitry with one or more processors.

Although the systems and methods may be described herein from time totime in the context of this example system, this is only one examplewith which these systems and methods may be implemented. After readingthis description, one of ordinary skill in the art will understand howthe systems and methods described herein can be implemented with thisand other scanners, microscopes and other imaging systems.

Implementations of the technology disclosed herein provide modularoptical analytic systems and methods. FIG. 1B is a perspective viewdiagram illustrating an example modular optical analytic system 180.System 180 may include a plurality of modular subassemblies. Forexample, in some implementations, system 180 comprises four subassemblymodules: line generation module (LGM) 182, focus tracking module (FTM)184, camera module (CAM) 186, and emission optical module (EOM) 188. Asused herein in the context of the LGM, FTM, EOM, or CAM, a module refersto a hardware unit (e.g., a modular subassembly).

In some implementations, LGM 182 may include one or more light sources.In some implementations, the one or more light sources may includecoherent light sources, such as laser diodes. In some examples, LGM 182may include a first light source configured to emit light in redwavelengths, and a second light source configured to emit light in greenwavelengths. LGM 182 may further include optical components, such asfocusing surfaces, lenses, reflective surfaces, or mirrors. The opticalcomponents may be positioned within an enclosure of LGM 182 as to directand focus the light emitted from the one or more light sources into anadjacent modular subassembly. One or more of the optical components ofLGM 182 may also be configured to shape the light emitted from the oneor more light sources into desired patterns. For example, in someimplementations, the optical components may shape the light into linepatterns (e.g., by using one or more Powell lenses, or other beamshaping lenses, diffractive or scattering components). One or more ofthe optical components may be located in one or more of the othermodular subassemblies. One or more of the modular subassemblies may alsoinclude one or more field replaceable sub-components. For example, LGM182 may include one or more laser modules which may be individuallyremoved from LGM 182 and replaced.

In some examples, the adjacent modular subassembly (coupled to LGM 182)may be EOM 188. Light from the one or more light sources of LGM 182 maybe directed out of LGM 182 and into EOM 188 through an interface baffleattached to LGM 182 and/or EOM 188. For example, the interface bafflemay be an aperture shaped to enable light to pass through its center,while obscuring interference from external light sources. EOM 188 mayalso include an objective, a tube lens, and or other optical componentsconfigured to shape, direct, and/or focus fluorescent light excited bythe one or more light sources of LGM 182.

Light passing through EOM 188 may be directed into one of the otheradjacent modular subassemblies, for example, CAM 186, through aninterface port. CAM 186 may include one or more light sensors. In someimplementations, a first light sensor may be configured to detect lightfrom the first light source of LGM 182 (e.g., in a red wavelength), anda second light sensor may be configured to detect light from the secondlight source of LGM 182 (e.g., a green wavelength). The light sensors ofCAM 186 may be positioned within an enclosure in a configuration such asto detect light from two incident light beams wherein the incident lightbeams may be spaced apart by a predetermined distance (e.g., between 1mm and 10 mm) based on the pitch of the two sensors. In some examples,the first light sensor and the second light sensor may be spaced apartfrom each other by between 3 mm and 8 mm. The light sensors may have adetection surface sufficiently sized to allow for beam drift, forexample, due to thermal effects or mechanical creep. Output data fromthe light sensors of CAM 186 may be communicated to a computerprocessor. The computer processor may then implement computer softwareprogram instructions to analyze the data and report or display thecharacteristics of the beam (e.g., focus, shape, intensity, power,brightness, position) to a graphical user interface (GUI), and/orautomatically control actuators and laser output to optimize the laserbeam. Beam shape and position may be optimized by actuating internaloptics of system 180 (e.g., tilting mirrors, articulating lenses, etc.).

FTM 184 may also couple to EOM 188 through an interface port. FTM 184may include instruments to detect and analyze the alignment and focus ofall of the optical components in system 180. For example, FTM 184 mayinclude a light source (e.g., a laser), optics, and a light sensor, suchas a digital camera or CMOS chip. The laser may be configured totransmit light source and optics may be configured to direct lightthrough optical components in system 180 and the light sensor may beconfigured to detect light being transmitted through optical componentsin system 180 and output data to a computer processor. The computerprocessor may then implement computer software program instructions toanalyze the data and report or display the characteristics of the laserbeam (e.g., focus, intensity, power, brightness, position) to agraphical user interface (GUI), and/or automatically control actuatorsand laser output to optimize the laser beam. In some examples, FTM 184may include a cooling system, such as an air or liquid cooling system asknown in the art.

In some implementations, LGM 182 may include light sources that operateat higher powers to also accommodate for faster scanning speeds (e.g.,the lasers in LGM 182 may operate at a five times greater power output).Similarly, the light source of laser module may operate at a higheroutput power and/or may also include a high resolution optical sensor toachieve nanometer scale focus precision to accommodate for fasterscanning speeds. The cooling system of FTM 184 may be enhanced toaccommodate the additional heat output from the higher powered laserusing cooling techniques known in the art.

In one example, each modular subassembly may mechanically couple to oneor more other modular subassemblies, and/or to a precision mountingplate 190. In some implementations, precision mounting plate 190 maymechanically couple to a stage assembly 192. Stage assembly 192 mayinclude motion dampers, actuators to actuate one or more componentswithin one or more modular subassemblies, cooling systems, and/or otherelectronics or mechanical components as known in the art.

The modular subassemblies may be prefabricated, configured, andinternally aligned. In some implementations, a control unit may beelectronically coupled to stage assembly 192 and communicatively coupledto a user interface to enable automatic or remote manual alignment ofone or more modular subassemblies after they have been coupled toprecision mounting plate 190. Each modular subassembly may be a fieldreplaceable unit (FRU), such that it may be removed from precisionmounting plate 190 and replaced with another functionally equivalentmodular subassembly without disturbing the alignment or configuration ofthe other modular subassemblies in the system.

Each module is pre-aligned and pre-qualified before integration intosystem 180. For example, assembly and configuration of LGM 182 mayinclude the mechanical coupling of one or more lasers or laser diodesinto an enclosure, and installation of control electronics to operatethe lasers or laser diodes. The entire LGM 182 may then be mounted on atest bed and operated to align the laser diodes within the enclosure, aswell as any optics or other components. The LGM enclosure may includeexternal mounting structures, such as mounting pins, datum, notches,tabs, slots, ridges, or other protrusions or indentations configured toalign the LGM 182 to the test bed, as well as to precision mountingplate 190 when installed in system 180. Once LGM 182 is configured andtested, it may be either installed in a system 180, or packaged andstored or shipped as a field replaceable unit (FRU).

Other modular subassemblies, such as FTM 184, CAM 186, or EOM 188, maybe similarly assembled, configured, and tested prior to installation onsystem 180. Each modular subassembly may be assembled using mechanicalcoupling methods to limit mobility of internal components within thesubassembly as desired. For example, components may be locked in placewith fasteners or welds to stop mobility of once the component isaligned to the other components or the enclosure of the modularsubassembly. Some components, as desired, may be coupled witharticulating joints or allowed to move within an enclosure such thattheir relative orientation may be adjusted after installation onprecision mounting plate 190. For example, each modular subassembly'srelative positioning may be controlled precisely using predeterminedmechanical tolerances (e.g., by aligning datum to receiving notches inan adjoining modular subassembly or in precision mounting plate 190)such as to enable overall optical alignment of system 180 with a limitednumber of adjustable degrees of freedom (e.g., fewer than 10 overalldegrees of freedom in some implementations).

FIG. 1C is a perspective view diagram illustrating an example precisionmounting plate 190. Precision mounting plate 190 may be fabricated fromlight weight, rigid, and heat tolerant materials. In someimplementations, precision mounting plate 190 may be fabricated from ametal (e.g., aluminum), ceramic, or other rigid materials as known inthe art. Precision mounting plate 190 may include precision alignmentstructures configured to mechanically couple to corresponding precisionalignment structures incorporated on the enclosures or housings of oneor more of the modular subassemblies. For example, precision alignmentstructures may include mounting pins, datums, tabs, slots, notches,grommets, magnets, ridges, indents, and/or other precision mountingstructures shaped to align a first surface (e.g., on precision mountingplate 190) to a second surface (e.g., an outer surface of the enclosureor housing of a modular subassembly. Referring to FIG. 1C, exampleprecision mounting plate 190 may include a plurality of LGM precisionmounting structures 194 configured to accept and mechanically couple tocorresponding precision mounting structures located on an outer surfaceof the enclosure of LGM 182. Similarly, precision mounting plate 190 mayinclude a plurality of EOM precision mounting structures 196 configuredto accept and mechanically couple to corresponding precision mountingstructures located on an outer surface of the enclosure of EOM 188. Bylocating LGM 182 and EOM 188 onto precision mounting plate 190 using theprecision mounting structures, LGM 182 and EOM 188 will align to eachother. Precision alignment structures located on the enclosures of othermodular subassemblies (e.g., FTM 184 and CAM 186) may then mechanicallycouple to respective precision alignment structures located on theenclosures of either LGM 182 or EOM 188, or on precision mounting plate190.

FIG. 1D illustrates a block diagram of an example modular opticalanalytic system. In some implementations, a modular optical analyticsystem may include an LGM 1182 with two light sources, 1650 and 1660,disposed therein. Light sources 1650 and 1660 may be laser diodes, diodepumped solid state lasers, or other light sources as known in the art,which output laser beams at different wavelengths (e.g., red or greenlight). The light beams output from laser sources 1650 and 1660 may bedirected through a beam shaping lens or lenses 1604. In someimplementations, a single light shaping lens may be used to shape thelight beams output from both light sources. In other implementations, aseparate beam shaping lens may be used for each light beam. In someexamples, the beam shaping lens is a Powell lens, such that the lightbeams are shaped into line patterns.

LGM 1182 may further include mirrors 1002 and 1004. A light beamgenerated by light source 1650 may reflect off 1002 as to be directedthrough an aperture or semi-reflective surface of mirror 1004, and intoEOM 1188 through a single interface port. Similarly, a light beamgenerated by light source 1660 may reflect off of mirror 1003 and mirror1004 as to be directed into EOM 1188 through a single interface port. Insome examples, an additional set of articulating mirrors may beincorporated adjacent to mirror 1004 to provide additional tuningsurfaces, for example, as illustrated in FIG. 1H.

Both light beams may be combined using dichroic mirror 1004. Both lightbeams may be directed through line forming optics, such as a Powelllens. Mirrors 1002 and 1004 may each be configured to articulate usingmanual or automated controls as to align the light beams from lightsources 1650 and 1660. The light beams may pass through a shutterelement 1006. EOM 1188 may include objective 1404 and a z-stage 1024which moves objective 1404 longitudinally closer to or further away froma target 1192. For example, target 1192 may include a liquid layer 1550and a translucent cover plate 1504, and a biological sample may belocated at an inside surface of the translucent cover plate as well aninside surface of the substrate layer located below the liquid layer.The z-stage may then move the objective as to focus the light beams ontoeither inside surface of the flowcell (e.g., focused on the biologicalsample). The biological sample may be DNA, RNA, proteins, or otherbiological materials responsive to optical sequencing as known in theart. In some implementations, the objective may be configured to focusthe light beams at a focal point beyond the flowcell, such as toincrease the line width of the light beams at the surfaces of theflowcell.

EOM 1188 may also include semi-reflective mirror 1020 to direct lightthrough objective 1404, while allowing light returned from target 1192to pass through. In some implementations, EOM 1188 may include a tubelens 1406 and a corrective lens 1450. Corrective lens 1450 may bearticulated longitudinally either closer to or further away fromobjective 1404 using a z-stage 1022 as to ensure accurate imaging, e.g.,to correct spherical aberration caused by moving objective 1404, and/orfrom imaging through a thicker substrate. Light transmitted throughcorrective lens 1450 and tube lens 1406 may then pass through filterelement 1012 and into CAM 1186. CAM 1186 may include one or more opticalsensors 1050 to detect light emitted from the biological sample inresponse to the incident light beams.

In some examples, EOM 1188 may further include semi-reflective mirror1018 to reflect a focus tracking light beam emitted from FTM 1184 ontotarget 1192, and then to reflect light returned from target 1192 backinto FTM 1184. FTM 1184 may include a focus tracking optical sensor todetect characteristics of the returned focus tracking light beam andgenerate a feedback signal to optimize focus of objective 1404 on target1192.

LGM 1182 is configured to generate a uniform line illumination throughan objective lens. For example, the objective lens may be located on EOM1188, or on an LGM alignment system used to align the internalcomponents of the LGM when the LGM is being assembled or maintained(e.g., and is physically separated from the modular optical analyticsystem). The LGM may use one or more Powell lenses to spread and/orshape the laser beams from single or near-single mode laser lightsources. Other beam shaping optics may be used to control uniformity andincrease tolerance such as an active beam expander, an attenuator, onerelay lenses, cylindrical lenses, actuated mirrors, diffractiveelements, and scattering components. Laser beams may intersect at theback focal point of objective lens to provide better tolerance onflowcell surfaces (e.g., as illustrated in FIG. 1.1). A Powell lens maybe located near the objective lens, or near a relay lens. The fan angleof the laser beam entering the imaging optics may be adjusted to matchthe field view of imaging optics.

The direction, size, and/or polarization of the laser beams may beadjusted by using lenses, mirrors, and/or polarizers. Optical lenses(e.g., cylindrical, spherical, or aspheric) may be used to activelyadjust the illumination focus on dual surfaces of the flowcell target.The light modules on LGM 1182 may be replaceable individually for fieldservice. LGM 1182 may include multiple units and each unit is designedfor particular/different wavelengths and polarization. Stacking multipleunits may be used to increase the laser power and wavelength options.Two or more laser wavelengths can be combined with dichroics andpolarizers.

To avoid photo-bleaching on adjacent area or photo-saturation offluorophores, illumination line profiles may be adjusted to fall withinpredetermined intensity ratio tolerances inside/outside imaging region.By widening the laser line patterns at the flowcell and/or sensor,higher scan speeds and laser powers may be employed (e.g., power andthroughput may be increased more than four-fold without experiencingphoto-saturation or photo-bleaching, or damaging the laser modules). Insome examples, laser power densities of more than 20 kW/cm² at theflowcell may over-saturate the fluorophores in the flowcell. When thisoccurs, the emission signal detected at the sensor will not increaselinearly with an increase in excitation power from the laser modules.

Methods for widening illumination lines using optics may include: addinga defocus lens, prism array, or diffuser after or before the Powelllens. In some implementations, these methods may also include reducingthe laser illumination beam size and/or reducing objective lens infiniteconjugation design. FIG. 1K illustrates a block diagram of an LGM andEOM system used to widen the laser line pattern on a flowcell to avoidphoto-saturation and photo-bleaching. The laser beam line width incidenton the flowcell may be increased to reduce excitation power density andavoid photo-saturation. Line width may be increased, for example, byincorporating a defocus lens, prism, array, or diffuser either in frontof or behind the Powell lens. In some implementations, the line widthmay be increased by defocusing the objective lens, as illustrated inFIG. 1K (e.g., moving the objective lens in the Z-axis) to focus theline pattern beyond the surfaces of the flowcell. In some examples,defocusing the line pattern to a distance of between about 50 micronsand about 150 microns from a distal surface of the flowcell may generatea line width larger than 10 microns, and effectively reducephoto-saturation and photo-bleaching effects.

When using a TDI sensor, the line width-to-beam intensity profile may bebalanced with signal-to-noise tolerances of the TDI sensor. For example,at very wide line widths, the signal-to-noise ratio may be too low to beeffective.

FIG. 1F illustrates a block diagram of a LGM alignment system. FIG. 1Gillustrates a perspective view of an LGM alignment system. Asillustrated, in some implementations, a green laser module may generatea first laser beam that reflects off two PZT mirrors. Similarly, a redlaser module may generate a second laser beam that also reflects off oftwo PZT mirrors and is combined with the first laser beam. Both laserbeams may then pass through a Powell lens to generate a line pattern,and then through a shutter, EOM optics, and an objective lens. In someimplementations, the laser beams may be defocused using a defocus lensprior to passing through the objective as to increase the line width ofthe laser beams. Alternatively, the laser beams may be defocused byarticulating the objective in the Z-axis. By focusing the laser beams ata focal point beyond the surfaces of the flowcell, the laser lines maybe widened as to disperse energy at the sample and avoidphoto-saturation, photo-bleaching, and laser damage at high scanningspeeds and high laser powers. In some implementations, the line patternsmay be increased in width from less than 5 microns to more than 13microns.

The LGM alignment system may include control surfaces to adjust ormanipulate relative positioning of mirrors 1002 and 1004, as well as thelenses, lasers, or other components or optics in the LGM. For example,adjustments may be made using manual manipulation of control knobs,screws, or other components. In other implementations, one or more ofthe optical components may be adjusted or manipulated automatically.Automatic control devices may include a motorized translation stage, anactuation device, one or more piezo stages, and/or one or more automaticswitch and flip mirrors and lenses. A software interface may be used tocontrol all the devices, test system, calibration, and test procedure.The alignment system includes a beam profiler (e.g., a 2D imagingsensor), imaging lens (replacing EOM objective lens), attenuator, and/oralignment targets. The software interface may be used to output reportsfor quality control and product evaluation. For example, the reports mayinclude data generated by the beam profiler relating to beam intensityand profile relative to each alignment configuration of the opticalcomponents of the LGM.

In some implementations, a method for aligning an LGM using an LGMalignment system may include identifying reasonable alignment positionsand tolerances for imaging optics, sensors, and mechanics relative to anLGM alignment system. The LGM alignment system is external to themodular optical analytic system. As such, the internal components of theLGM may be assembled and aligned prior to installation in the modularoptical analytic system. The internal components of the LGM may also bealigned during a maintenance activity.

In some implementations, alignment of the LGM optical components may beaccomplished using actuated devices for automatic tracking andadjustment during sequencing or between sequencing cycles/runs. Forexample, the actuated devices can be a piezo stage, a motorizedactuator, or similar devices known in the art. The actuated devices mayalso compensate for drift caused by temperature changes, as well asdecay of optical components including lasers, lens, and mounts.

Each optical component may mechanically couple to an enclosure oroptical frame using a mechanical interface with precision contact pads,dowel pins, stoppers, or other precision mechanical mounting surfaces asknown in the art.

FIGS. 2A and 2B are diagrams illustrating precision mounting structureson EOM 188. In several implementations, EOM 188 may include an EOMenclosure 210. EOM 188 may mechanically and optically couple to LGM 182,FTM 184, and CAM 186 (e.g., the enclosure of EOM 188 may include one ormore apertures corresponding to and aligned with an aperture located onan enclosure of each of the other modular subassemblies to enable light,generated by a light source(s) in LGM 182 and/or FTM 184 to transitthrough the apertures and internal optics of EOM 188.). As illustratedin FIG. 2B, EOM enclosure 210 may include FTM precision mountingstructures 212 configured to align and mechanically couple (e.g.,physically attach) to corresponding precision mounting structureslocated on an outer surface of an enclosure of FTM 184. Similarly, EOMenclosure 210 may include CAM mounting structures 222 configured toalign and mechanically couple to corresponding precision mountingstructures located on an outer surface of an enclosure 220 of CAM 186.

FIGS. 3A, 3B, and 3C are diagrams illustrating precision mountingstructures on FTM 184. Referring to FIG. 3A, FTM 184 may include a lightsource and optical sensors positioned within FTM enclosure 300. FTMenclosure 300 may include interface ports for electronic interfaces 302,304, and 306 to control the light source and optical sensors. FTMenclosure 300 may also include precision mounting structures 312 (e.g.,precision mounting feet configured to mechanically couple to recesses orpredetermined locations on precision mounting plate 190). FTM enclosure300 may further include precision mounting structures 314 configured toalign and mechanically couple to corresponding precision mountingstructures 212 located on an outer surface of EOM enclosure 210

Pre-assembling, configuring, aligning, and testing each modularsubassembly, and then mounting each to precision mounting plate 190 toassist in system alignment, may reduce the amount of post-installationalignment required to meet desired tolerances. In one example, postinstallation alignment between EOM 188 and each of the other subassemblymodules may be accomplished by interfacing corresponding module ports(e.g., an EOM/FTM port, an EOM/CAM port, and an EOM/LGM port), andaligning the modular subassemblies to each other by manually orautomatically articulating the position (in the X, Y, or Z axis), angle(in the X or Y direction), and the rotation of each modular subassembly.Some of the degrees of freedom may be limited by precision alignmentstructures that predetermine the position and orientation of the modularsubassembly with respect to precision mounting plate 190 and adjacentmodular subassemblies. Tuning and aligning the internal optics of system180 may then be accomplished by articulating components internal to themodular subassemblies (e.g., by tilting or moving in either X, Y, or Zmirrors and lenses).

FIG. 4A is a side view diagram illustrating an example modular opticalanalytic system. As illustrated in FIG. 4A, LGM 182 and EOM 188 may bealigned and mechanically coupled to precision mounting plate 190, aswell as to each other. EOM 188 may include an objective 404 aligned, viamirror 408 with tube lens 406, which in turn is optically coupled to LGM182, such that light beams generated by LGM 182 transmit through aninterface baffle between LGM 182 and EOM 188, pass through objective404, and strike an optical target. Responsive light radiation from thetarget may then pass back through objective 404 and into tube lens 406.Tube lens 406 may include a lens element 450 configured to articulatealong the z-axis to correct for spherical aberration artifactsintroduced by objective 404 imaging through varied thickness of flowcellsubstrate or cover glass. For example, FIGS. 4B and 4C are blockdiagrams illustrating different configurations of tube lens 406. Asillustrated, lens element 450 may be articulated closer to or furtheraway from objective 404 to adjust the beam shape and path.

In some implementations, EOM 188 may be mechanically coupled to az-stage, e.g., controlled by actuators on alignment stage 192. In someexamples, the z-stage may be articulated by a precision coil andactuated by a focusing mechanism which may adjust and moves objective404 to maintain focus on a flowcell. For example, the signal to controlto adjust the focus may be output from FTM 184. This z-stage may alignthe EOM optics, for example, by articulating objective 404, tube lens406, and/or lens element 450.

FIGS. 5A and 5B are diagrams illustrating FTM 184. FTM 184 may interfacewith EOM 188 through FTM/EOM interface port 502. As illustrated in FIG.5A, light beams originating in FTM 184 and passing through the optics ofEOM 188 may reflect off flowcell 504. As disclosed herein, FTM 184 maybe configured to provide feedback to a computer processor in order tocontrol alignment and positioning of optical components throughoutsystem 180. For example, FTM 184 may employ a focus mechanism using twoor more parallel light beams which pass through objective 404 andreflect off flowcell 504. Movement of the flowcell away from an optimalfocus position may cause the reflected beams to change angle as theyexit objective 404. That angle may be measured by an optical sensorlocated in FTM 184. In some examples, the distance of the light pathbetween the optical sensor surface and the objective 404 may be between300 mm and 700 mm distance. FTM 184 may initiate a feedback loop usingan output signal from the optical sensor to maintain a pre-determinedlateral separation between beam spot patterns of the two or moreparallel light beams by adjusting the position of objective 404 usingthe z-stage in the EOM.

Some implementations of system 180 provide a compensation method for topand bottom surface imaging of flowcell 504. In some examples, flowcell504 may include a cover glass layered on a layer of liquid and asubstrate. For example, the cover glass may be between about 100 um andabout 500 um thick, the liquid layer may be between about 50 um andabout 150 um thick, and the substrate may be between about 0.5 and about1.5 mm thick. In one example, a DNA sample may be introduced at the topand bottom of the liquid channel (e.g., at the top of the substrate, andbottom of the cover glass). To analyze the sample, the focal point ofthe incident light beams at various depths of flowcell 504 may beadjusted by moving the z-stage (e.g., to focus on the top of thesubstrate or the bottom of the cover glass. Movement of objective 404 tochange incident beam focal points within flowcell 504 may introduceimaging artifacts or defects, such as spherical aberration. To correctfor these artifacts or defects, lens element 450 within tube lens 406may be moved closer to or further away from objective 404.

In some examples FTM 184 may be configured as a single FRU with noreplaceable internal components. To increase longevity and reliabilityof FTM internal components, such as the laser, laser output may bereduced (for example, below 5 mW).

FIGS. 6 and 7 are diagrams illustrating LGM 182 and EOM 188. Asillustrated, LGM 182 may interface with EOM 188 through LGM/EOMinterface baffle 602. LGM 182 is a photon source for system 180. One ormore light sources (e.g., light sources 650 and 660) may be positionedwithin an enclosure of LGM 182. Light generated from light sources 650and 660 may be directed through a beam shaping lens 604 and into theoptical path of EOM 188 through LGM/EOM interface baffle 602. Forexample, light source 650 may be a green laser and light source 660 maybe a red laser. The lasers may operate at high powers (e.g., more than 3Watts). One or more beam shaping lenses 604 may be implemented to shapethe light beams generated from the light sources into desired shapes(e.g., a line).

Photons generated by light sources 650 and 660 (e.g., green wavelengthphotons and red wavelength photons) may excite fluorophores in DNAlocated on flowcell 504 to enable analysis of the base pairs presentwithin the DNA. High speed sequencing employs high velocity scanning todeliver a sufficient photon dose to the DNA fluorophores, to stimulatesufficient emission of reactive photons from the DNA sample to bedetected by the light sensors in CAM 186.

Beam shaping lens 604 may be a Powell lens that spreads the Gaussianlight emitted by lasers 650 and 660 into a uniform profile (inlongitudinal direction), which resembles a line. In some exampleimplementations, a single beam shaping 604 lens may be used for multiplelight beams (e.g., both a red and a green light beam) which may beincident on the front of beam shaping lens 604 at differentpre-determined angles (e.g., plus or minus a fraction of a degree) togenerate a separate line of laser light for each incident laser beam.The lines of light may be separated by a pre-determined distance toenable clear detection of separate signals, corresponding to each lightbeam, by the multiple optical sensors in CAM 186. For example, a greenlight beam may ultimately be incident on a first optical sensor in CAM186 and a second light beam may ultimately be incident on a secondoptical sensor in CAM 186.

In some examples, the red and green light beams may becoincident/superimposed as they enter beam shaping lens 604 and thenbegin to fan out into respective line shapes as they reach objective404. The position of the beam shaping lens may be controlled with tighttolerance near or in close proximity to light sources 650 and 660 tocontrol beam divergence and optimize shaping of the light beams, i.e.,by providing sufficient beam shape (e.g., length of the line projectedby the light beam) while still enabling the entire beam shape to passthrough objective 404 without clipping any light. In some examples,distance between beam shaping lens 604 and objective 404 is less thanabout 150 mm.

In some implementations, system 180 may further comprise a modularsubassembly having a pocket to receive the optical target. The body maycomprise aluminum that includes a pigment having a reflectivity of nomore than about 6.0%. The body may include an inset region located atthe top surface and surrounding the pocket. The modular subassembly mayfurther comprise a transparent grating layer mounted in the inset regionand may be positioned above the optical target and spaced apart from theoptical target by a fringe gap. The body may include a pocket to receivethe optical target. The body may include a diffusion well located belowthe optical target. The diffusion well may receive excitation lightpassing through the optical target. The diffusion well may include awell bottom having a pigment based finish that exhibits a reflectivelyof no more than about 6.0%.

One of the modular subassemblies of system 180 may further include anoptical detection device. Objective 404 may emit excitation light towardthe optical target and receive fluorescence emission from the opticaltarget. An actuator may be configured to position objective 404 to aregion of interest proximate to the optical target. The processor maythen execute program instructions for detecting fluorescence emissionfrom the optical target in connection with at least one of opticalalignment and calibration of an instrument.

In some examples, objective 404 may direct excitation light onto theoptical target. The processor may derive reference information from thefluorescence emission. The processor may utilize the referenceinformation in connection with the at least one of optical alignment andcalibration of the instrument. The optical target may be permanentlymounted at a calibration location proximate to objective 404. Thecalibration location may be separate from flowcell 504. The solid bodymay represent a substrate comprising a solid host material with thefluorescing material embedded in the host material. The solid body mayrepresent at least one of an epoxy or polymer that encloses quantum dotsthat emit fluorescence in one or more predetermined emission bands ofinterest when irradiated by the excitation light.

FIG. 8 is a diagram illustrating an example process for installing andconfiguring a modular optical analytic system 800. Process 800 mayinclude positioning a plurality of light sources and a beam shaping lenswithin a first subassembly at step 805. For example, the plurality oflight sources may include light source 650 and light source 660. Thefirst subassembly may be an LGM, which may include an LGM enclosure towhich the light sources are mounted and aligned. The beam shaping lensmay be a Powell lens, also mounted within the LGM enclosure, andconfigured to shape light beams generated by light sources 650 and 660into separate line patterns.

Process 800 may also include positioning a tube lens and objectivewithin a second subassembly at step 815. For example, the secondsubassembly may be an EOM and may include an EOM enclosure to which theobjective and tube lens are mounted and aligned.

Process 800 may also include positioning a plurality of optical sensorswithin a third subassembly at step 825. For example, the thirdsubassembly may be a CAM and may include a CAM enclosure to which theoptical sensors are aligned and mounted. There may be a correspondingoptical sensor to each light source from step 805.

Process 800 may also include positioning a focus tracking light sourceand optical sensor within a fourth subassembly at step 835. For example,the fourth subassembly may be an FTM and may include an FTM enclosure towhich the focus tracking light source and optical sensor are mounted.

In some implementations, process 800 may further include individuallytesting each subassembly at step 845. For example, testing may includeprecisely tuning and/or aligning the internal components of eachsubassembly to the subassembly's enclosure. Each subassembly may then bemechanically coupled to a precision mounting plate at step 855. Forexample, the precision mounting plate may be precision mounting plate190. The entire system may then be aligned and tuned by powering thefocus tracking light source in the fourth subassembly and capturing anoutput signal from the focus tracking optical sensor of the fourthsubassembly to find an optimal focus of the optical target. The outputsignal from the target may be input into a computer processor configuredto analyze the characteristics of light beams generated by the focustracking light source, and then provide feedback to actuators on one ormore of the subassemblies, or to a graphical user interface to enabletuning of the optical components to optimize beam shape, power, andfocus.

As noted above, in various implementations an actuator can be used toposition the sample stage relative to the optical stage by repositioningeither the sample stage or the optical stage (or parts thereof), or bothto achieve the desired focus setting. In some implementations,piezoelectric actuators can be used to move the desired stage. In otherimplementations, a voice coil actuator can be used to move the desiredstage. In some applications, the use of a voice coil actuator canprovide reduced focusing latency as compared to its piezoelectriccounterparts. For implementations using a voice coil actuator, coil sizemay be chosen as a minimum coil size needed to provide the desiredmovement such that the inductance in the coil can also be minimized.Limiting coil size, and therefore limiting its inductance, providesquicker reaction times and requires less voltage to drive the actuator.

As described above, regardless of the actuator used, focus informationfrom points other than a current sample location can be used todetermine the slope or the magnitude of change in the focus setting forscanning operations. This information can be used to determine whetherto feed the drive signal to the actuator earlier and how to set theparameters of the drive signal. Additionally, in some implementationsthe system can be pre-calibrated to allow drive thresholds to bedetermined for the actuator. For example, the system can be configuredto supply to the actuator drive signals at different levels of controloutput to determine the highest amount of control output (e.g., themaximum amount of drive current) the actuator can withstand withoutgoing unstable. This can allow the system to determine a maximum controloutput amount to be applied to the actuator.

As used herein, the term engine may describe a given unit offunctionality that can be performed in accordance with one or moreimplementations of the technology disclosed herein. As used herein, anengine may be implemented utilizing any form of hardware, software, or acombination thereof. For example, one or more processors, controllers,ASICs, PLAs, PALs, CPLDs, FPGAs, logical components, software routinesor other mechanisms may be implemented to make up an engine. Inimplementation, the various engines described herein may be implementedas discrete engines or the functions and features described can beshared in part or in total among one or more engines. In other words, aswould be apparent to one of ordinary skill in the art after reading thisdescription, the various features and functionality described herein maybe implemented in any given application and can be implemented in one ormore separate or shared engines in various combinations andpermutations. Even though various features or elements of functionalitymay be individually described or claimed as separate engines, one ofordinary skill in the art will understand that these features andfunctionality can be shared among one or more common software andhardware elements, and such description shall not require or imply thatseparate hardware or software components are used to implement suchfeatures or functionality.

Where components or engines of the technology are implemented in wholeor in part using software, in one implementation, these softwareelements can be implemented to operate with a computing or processingengine capable of carrying out the functionality described with respectthereto. One such example computing engine is shown in FIG. 9. Variousimplementations are described in terms of this example computing engine900. After reading this description, it will become apparent to a personskilled in the relevant art how to implement the technology using othercomputing engines or architectures.

Referring now to FIG. 9, computing engine 900 may represent, forexample, computing or processing capabilities found within desktop,laptop and notebook computers; hand-held computing devices (PDA's, smartphones, cell phones, palmtops, etc.); mainframes, supercomputers,workstations or servers; or any other type of special-purpose orgeneral-purpose computing devices as may be desirable or appropriate fora given application or environment. Computing engine 900 may alsorepresent computing capabilities embedded within or otherwise availableto a given device. For example, a computing engine may be found in otherelectronic devices such as, for example, digital cameras, navigationsystems, cellular telephones, portable computing devices, modems,routers, WAPs, terminals and other electronic devices that may includesome form of processing capability.

Computing engine 900 may include, for example, one or more processors,controllers, control engines, or other processing devices, such as aprocessor 904. Processor 904 may be implemented using a general-purposeor special-purpose processing engine such as, for example, amicroprocessor, controller, or other control logic. In the illustratedexample, processor 904 is connected to a bus 902, although anycommunication medium can be used to facilitate interaction with othercomponents of computing engine 900 or to communicate externally.

Computing engine 900 may also include one or more memory engines, simplyreferred to herein as main memory 908. For example, preferably randomaccess memory (RAM) or other dynamic memory, may be used for storinginformation and instructions to be executed by processor 904. Mainmemory 908 may also be used for storing temporary variables or otherintermediate information during execution of instructions to be executedby processor 904. Computing engine 900 may likewise include a read onlymemory (“ROM”) or other static storage device coupled to bus 902 forstoring static information and instructions for processor 904.

The computing engine 900 may also include one or more various forms ofinformation storage mechanism 910, which may include, for example, amedia drive 912 and a storage unit interface 920. The media drive 912may include a drive or other mechanism to support fixed or removablestorage media 914. For example, a hard disk drive, a floppy disk drive,a magnetic tape drive, an optical disk drive, a CD or DVD drive (R orRW), or other removable or fixed media drive may be provided.Accordingly, storage media 914 may include, for example, a hard disk, afloppy disk, magnetic tape, cartridge, optical disk, a CD or DVD, orother fixed or removable medium that is read by, written to or accessedby media drive 912. As these examples illustrate, the storage media 914can include a computer usable storage medium having stored thereincomputer software or data.

In alternative implementations, information storage mechanism 910 mayinclude other similar instrumentalities for allowing computer programsor other instructions or data to be loaded into computing engine 900.Such instrumentalities may include, for example, a fixed or removablestorage unit 922 and an interface 920. Examples of such storage units922 and interfaces 920 can include a program cartridge and cartridgeinterface, a removable memory (for example, a flash memory or otherremovable memory engine) and memory slot, a PCMCIA slot and card, andother fixed or removable storage units 922 and interfaces 920 that allowsoftware and data to be transferred from the storage unit 922 tocomputing engine 900.

Computing engine 900 may also include a communications interface 924.Communications interface 924 may be used to allow software and data tobe transferred between computing engine 900 and external devices.Examples of communications interface 924 may include a modem orsoftmodem, a network interface (such as an Ethernet, network interfacecard, WiMedia, IEEE 802.XX or other interface), a communications port(such as for example, a USB port, IR port, RS232 port Bluetooth®interface, or other port), or other communications interface. Softwareand data transferred via communications interface 924 may be carried onsignals, which can be electronic, electromagnetic (which includesoptical) or other signals capable of being exchanged by a givencommunications interface 924. These signals may be provided tocommunications interface 924 via a channel 928. This channel 928 maycarry signals and may be implemented using a wired or wirelesscommunication medium. Some examples of a channel may include a phoneline, a cellular link, an RF link, an optical link, a network interface,a local or wide area network, and other wired or wireless communicationschannels.

In this document, the terms “computer program medium” and “computerusable medium” are used to generally refer to media such as, forexample, memory 908, storage unit 922, media 914, and channel 928. Theseand other various forms of computer program media or computer usablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processing device for execution. Such instructionsembodied on the medium, are generally referred to as “computer programcode” or a “computer program product” (which may be grouped in the formof computer programs or other groupings). When executed, suchinstructions may enable the computing engine 900 to perform features orfunctions of the disclosed technology as discussed herein.

While various implementations of the disclosed technology have beendescribed above, it should be understood that they have been presentedby way of example only, and not of limitation. Likewise, the variousdiagrams may depict an example architectural or other configuration forthe disclosed technology, which is done to aid in understanding thefeatures and functionality that can be included in the disclosedtechnology. The disclosed technology is not restricted to theillustrated example architectures or configurations, but the desiredfeatures can be implemented using a variety of alternative architecturesand configurations. Indeed, it will be apparent to one of skill in theart how alternative functional, logical or physical partitioning andconfigurations can be implemented to implement the desired features ofthe technology disclosed herein. Also, a multitude of differentconstituent engine names other than those depicted herein can be appliedto the various partitions. Additionally, with regard to flow diagrams,operational descriptions and method claims, the order in which the stepsare presented herein shall not mandate that various implementations beimplemented to perform the recited functionality in the same orderunless the context dictates otherwise.

It should be appreciated that all combinations of the foregoing concepts(provided such concepts are not mutually inconsistent) are contemplatedas being part of the inventive subject matter disclosed herein. Inparticular, all combinations of claimed subject matter appearing at theend of this disclosure are contemplated as being part of the inventivesubject matter disclosed herein. For example, although the disclosedtechnology is described above in terms of various exampleimplementations, it should be understood that the various features,aspects and functionality described in one or more of the individualimplementations are not limited in their applicability to the particularimplementation with which they are described, but instead can beapplied, alone or in various combinations, to one or more of the otherimplementations of the disclosed technology, whether or not suchimplementations are described and whether or not such features arepresented as being a part of a described implementation. Thus, thebreadth and scope of the technology disclosed herein should not belimited by any of the above-described example implementations.

Terms and phrases used in this document, and variations thereof, unlessotherwise expressly stated, should be construed as open ended as opposedto limiting. As examples of the foregoing: the term “including” shouldbe read as meaning “including, without limitation” or the like; the term“example” is used to provide example instances of the item indiscussion, not an exhaustive or limiting list thereof; the terms “a” or“an” should be read as meaning “at least one,” “one or more” or thelike; and adjectives such as “conventional,” “traditional,” “normal,”“standard,” “known” and terms of similar meaning should not be construedas limiting the item described to a given time period or to an itemavailable as of a given time, but instead should be read to encompassconventional, traditional, normal, or standard technologies that may beavailable or known now or at any time in the future. Likewise, wherethis document refers to technologies that would be apparent or known toone of ordinary skill in the art, such technologies encompass thoseapparent or known to the skilled artisan now or at any time in thefuture.

The terms “substantially” and “about” used throughout this disclosure,including the claims, are used to describe and account for smallfluctuations, such as due to variations in processing. For example, theycan refer to less than or equal to ±5%, such as less than or equal to±2%, such as less than or equal to ±1%, such as less than or equal to±0.5%, such as less than or equal to ±0.2%, such as less than or equalto ±0.1%, such as less than or equal to ±0.05%.

To the extent applicable, the terms “first,” “second,” “third,” etc.herein are merely employed to show the respective objects described bythese terms as separate entities and are not meant to connote a sense ofchronological order, unless stated explicitly otherwise herein.

The term “coupled” refers to direct or indirect joining, connecting,fastening, contacting or linking, and may refer to various forms ofcoupling such as physical, optical, electrical, fluidic, mechanical,chemical, magnetic, electromagnetic, communicative or other coupling, ora combination of the foregoing. Where one form of coupling is specified,this does not imply that other forms of coupling are excluded. Forexample, one component physically coupled to another component mayreference physical attachment of or contact between the two components(directly or indirectly), but does not exclude other forms of couplingbetween the components such as, for example, a communications link(e.g., an RF or optical link) also communicatively coupling the twocomponents. Likewise, the various terms themselves are not intended tobe mutually exclusive. For example, a fluidic coupling, magneticcoupling or a mechanical coupling, among others, may be a form ofphysical coupling.

The presence of broadening words and phrases such as “one or more,” “atleast,” “but not limited to” or other like phrases in some instancesshall not be read to mean that the narrower case is intended or requiredin instances where such broadening phrases may be absent. The use of theterm “engine” does not imply that the components or functionalitydescribed or claimed as part of the engine are all configured in acommon package. Indeed, any or all of the various components of anengine, whether control logic or other components, can be combined in asingle package or separately maintained and can further be distributedin multiple groupings or packages or across multiple locations.

Additionally, the various implementations set forth herein are describedin terms of exemplary block diagrams, flow charts and otherillustrations. As will become apparent to one of ordinary skill in theart after reading this document, the illustrated implementations andtheir various alternatives can be implemented without confinement to theillustrated examples. For example, block diagrams and their accompanyingdescription should not be construed as mandating a particulararchitecture or configuration.

1. An imaging system, comprising: a line generation module and anobjective lens; the line generation module comprising: a first lightsource to emit a first light beam at a first wavelength; a second lightsource to emit a second light beam at a second wavelength; and one ormore line forming optics to shape a light beam emitted by the firstlight source into a line and a light beam emitted by the second lightsource into a line; and wherein the objective lens is to focus the firstlight beam and the second light beam at a focal point external to asample of a sampling structure.
 2. The imaging system of claim 1,wherein the sampling structure comprises a cover plate, a substrate, anda liquid passage between the cover plate and substrate, wherein theliquid passage comprises a top interior surface and a bottom interiorsurface, wherein the sample is located at the top interior surface or atthe bottom interior surface of the liquid passage.
 3. The imaging systemof claim 2, wherein the focal point is below the bottom interior surfaceof the liquid passage to increase a line width of the first light beamand a line width of the second line beam at the top interior surface ofthe sampling structure.
 4. The imaging system of claim 2, wherein thefocal point is above the bottom interior surface of the liquid passageas to increase a line width of the first light beam and a line width ofthe second line beam at the top interior surface of the samplingstructure.
 5. The imaging system of claim 3, wherein the focal point isbetween about 50 μm and about 150 μm below the bottom interior surfaceof the sampling structure.
 6. The imaging system of claim 4, wherein thefocal point is between about 50 μm and about 150 μm above the bottominterior surface of the sampling structure.
 7. The imaging system ofclaim 2, further comprising: a time delay integration (TDI) sensor todetect fluorescence emissions from the sample, wherein the TDI sensorhas a pixel size between about 5 μm and about 15 μm, a sensor widthbetween about 0.4 mm and about 0.8 mm, and a sensor length between about16 mm and about 48 mm.
 8. The imaging system of claim 2, wherein theline width of the first light beam and the line width of the secondlight beam is between about 10 μm and about 30 μm.
 9. The imaging systemof claim 5, wherein the line length of the first light beam and the linelength of the second light is between about 1 mm and about 1.5 mm. 10.The imaging system of claim 6, wherein the line length of the firstlight beam and the line length of the second light is between about 1 mmand about 1.5 mm.
 11. The imaging system of claim 2, further comprisinga one or more line widening optics to increase the line width of thefirst light beam and the line width of the second light beam.
 12. Theimaging system of claim 11, wherein the one or more line widening opticscomprises defocus lens, a prism, or a diffuser.
 13. The imaging systemof claim 11, wherein the one or more line widening optics comprises aPowell lens positioned after a defocus lens in an optical path from thefirst light source and second light source to the objective lens. 14.The imaging system of claim 2, wherein the line width of the first lightbeam is increased to lower overall power density of the first light beamon a surface of the sample such that the power density of the firstlight beam on the surface of the sample is below a photosaturationthreshold of a first dye on the sample, and wherein the line width ofthe second light beam is increased to lower overall power density of thesecond light beam on a surface of the sample such that the power densityof the second light beam on the surface of the sample is below aphotosaturation threshold of a second dye on the sample.
 15. The imagingsystem of claim 7, further comprising: a z-stage for articulating theobjective to adjust the line width of the first light beam and to adjustthe line width of the second light beam.
 16. The imaging system of claim15, further comprising: a processor; and a non-transitory computerreadable medium with computer executable instructions embedded thereon,the computer executable instructions configured to cause the imagingsystem to: determine a quality of a signal from the TDI sensor; andarticulate the objective in the z-axis to adjust the focal point andoptimize the quality of the signal from the TDI sensor.
 17. A DNAsequencing system, comprising: a line generation module and an objectivelens; the line generation module comprising: a plurality of lightsources, each light source to emit a light beam; and one or more lineforming optics to shape each light beam into a line; and wherein theobjective lens or the one or more line forming optics are to increase awidth of each line at a first surface or a second surface of a flowcell.18. The system of claim 17, wherein the objective lens is to focus eachlight beam at a focal point external to an interior surface of theflowcell as to increase the width of each line at the first surface orthe second surface of the flowcell.
 19. The system of claim 18, whereinthe focal point is above a top interior surface of the flowcell or belowa bottom interior surface of the flowcell.
 20. The system of claim 19,wherein the focal point is between about 50 μm and about 150 μm belowthe bottom interior surface of the flowcell or between about 50 μm andabout 150 μm above the top interior surface of the flowcell.